PROJECT SUMMARY This era may come to be remembered as one in which infectious diseases made a dramatic worldwide resurgence, owing to the rise of antibiotic resistance and the limited number of antibiotics in the drug development pipeline. New antibiotics are needed and the emergence of resistance to almost any antibiotic underscores the need to understand the molecular details of antibiotic modes of action to help guide the development of new antibiotics. Gram-positive bacteria surround themselves with a thick cell wall that is essential to cell survival and is a major target of antibiotics. Quantifying alterations in cell-wall composition are crucial to evaluating drug modes of action, particularly important for human pathogens that are now resistant to multiple antibiotics such as Staphylococcus aureus. Yet, as a heterogeneous insoluble polymeric matrix, the cell wall poses a challenge to non-perturbative analyses of composition and architecture. Solid-state NMR has emerged as a powerful tool to measure parameters of composition and architecture in the context of intact cell walls and whole cells and was employed to define the mode of action of vancomycin analogs including oritvancin (OrbactivTM), focusing on specifically labeled samples and two key peptidoglycan sites (crosslinks and bridgelinks). Analyses of unlabeled or uniformly-labeled samples report more broadly on overall carbon and nitrogen composition in cell walls and whole cells, can be readily extended to other organisms, do not require considerations of label scrambling and dilution, and can identify alterations due to different classes of antibiotics. In this project, we will introduce new protocols, integrate these with existing approaches, and build and describe an effective solid-state NMR toolkit to define and quantify bacterial cell-wall composition, including peptidoglycan and teichoic acid components, in intact preparations of cell walls and whole cells. These approaches will integrate different labeling strategies coupled with appropriate NMR pulse sequences. The results from these approaches complement extensive biochemical data usually already available for old and new antibiotics and will be uniquely enabling in their ability to identify and quantitatively compare cell-wall components in intact samples without degradative hydrolyses and perturbations that limit quantitative comparisons in solution-based assays, particularly important for Gram-positive organisms with cell walls that resist complete dissolution. The methods will be tested with a panel of established antibiotics and will be used to investigate different bacterial strains and the modes of action of four new antibiotics.